La photorespiration est un processus essential chez tous les organismes photosynthétiques. Elle est déclenchée par l’activité oxygénase de la Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO) menant à la production d’une molécule de 3-phosphoglycerate and une molécule de 2-phosphoglycolate (2PG). Le 2PG est toxique et sera recyclé par la photorespiration qui implique huit principales enzymes et prend place dans les chloroplastes, les peroxysomes, les mitochondries et le cytosol. Bien que la photorespiration aboutisse à une efficacité réduite de l’assimilation du CO₂ photosynthétique et soit considérée comme un processus inutile, le phénotype de croissance des mutants d’enzymes photorespiratoires (croissance réduite, chlorose) reflète l’importance de ce processus dans la croissance et le développement normal car il interagit avec plusieurs voies métaboliques primaires. Les données actuelles montrent que sept des huit principales enzymes photorespiratoires pourraient être phosphorylées et qu’ainsi la phosphorylation pourrait être un élément régulateur essentiel du cycle photorespiratoire. Afin de mieux comprendre la régulation du cycle photorespiratoire, nous avons étudié l’effet d’une phosphorylation/ absence de phosphorylation sur la sérine hydroxyméthyltransférase 1 mitochondriale (SHMT1) et de l’hydroxypyruvate réductase peroxisomale en utilisant des versions de ces enzymes mimant une phosphorylation (sérine ou la thréonine mutée en acide aspartique) ou une absence de phosphoryaltion (sérine ou thréonine mutée en alanine).Deux sites sont phosphorylés chez HPR1: S229 et T335. La mutation de ces sites montre que seule la version mimant une phosphorylation sur le site T335 (HPR1 T335D) entraîne une activité réduite de la protéine recombinante HPR1. Ce résultat a été confirmé in vivo puisque le mutant Arabidopsis hpr1 exprimant HPR1 T33D était incapable de totalement complémenter le phénotype photorespiratoire du mutant hpr1.Par complémentation du mutant d’Arabidopsis shm1-1 par une forme sauvage de SHMT1, d’une version mimant (S31D) ou non (S31A) une phosphorylation, les résultats ont montré que toutes les formes de SHMT1 pouvaient presque totalement complémenter le phénotype de croissance de shm1-1. Cependant, chaque ligne transgénique n'avait que 50% de l'activité de SHMT normale. En réponse à un stress dû au sel ou à la sécheresse, les lignées Compl-S31D ont montré un déficit de croissance plus accentué que les autres lignées transgéniques. Cette sensibilité au sel semble refléter les quantités réduites de protéines SHMT1-S31D ainsi qu’une activité plus faible ayant un impact sur le métabolisme des feuilles, entraînant une sous-accumulation de proline et une suraccumulation de polyamines. La mutation S31D de la protéine SHMT1 a également entraîné une réduction de la fermeture stomatique induite par le sel et l'ABA. Ainsi, nos résultats soulignent l’importance du maintien de l’activité du SHMT1 photorespiratoire dans des conditions de stress dû au sel et à la sécheresse et indiquent que la phosphorylation de SHMT1 S31 pourrait être impliquée dans la modulation de la stabilité de la protéine SHMT1. / Photorespiration is an essential process in oxygenic photosynthetic organisms, and it is triggered by the oxygenase activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO) to produce one molecular 3-phosphoglycerate and one molecular 2-phosphoglycolate. The toxic 2-PG is recycled by the photorespiratory pathway which includes eight core enzymes and takes place in chloroplasts, peroxisomes and metochondria and cytosol. Although the photorespiration leads to a reduced efficiency of the photosynthetic CO₂ assimilation and thereby is considered as a wasteful process, the growth phenotype of the photorespiratory enzymes can reflect the importance of this process in normal growth and development of air-grown plants. Normally, for most photorespiratory enzyme mutants, they exhibit small, chlorotic plants sometimes non-viable in air which are not observed when the mutants are grown under high CO₂ condition that limit the photorespiration by reducing the RuBisCO oxygenase activity. Photorespiratory cycle interacts with several major primary metabolic pathways, thus is a highly regulated and extensive works. Current data show that seven of eight core photorespiratory enzymes could be phosphorylated and the protein phosphorylation seems to be a critical regulatory component of the photorespiratory cycle. In order to better understand the regulation of the photorespiratory cycle, we explored the effect of SHMT1 and HPR1 phosphorylation/non-phosphorylation events on plant physiology and metabolism by several methods: Site-directed mutagenesis assay, complementation assay, activity assay, stomatal aperture assays, plant salt/drought resistance assays, metabolites measurement, gas exchange measurement. The results show the phosphorylation mimicking version of HPR1 at T335 results to a less HPR1 activity and retarded growth at the ambient air condition. For the phosphorylation mimicking version of SHMT1 at S31 resulted in a less stability leading to a reduced resistance to drought and salt stress. The decline of resistance against abiotic stress was mainly due to impairment in the closure of stomata which were unable to respond properly to ABA probably because of a default in the PLC pathway. So there results indicate that the phosphorylation of SHTM1 leads to a negative effect for the plant growth especially under stress condition. Thus, we propose that the SHMT1 can be phosphorylated at a basic level under normal growth conditions, once the photorespiratory flux is increased such under salt stress condition, the SHMT1 should be dephosphorylated to stabilize SHMT1 and sustain a high photorespiration flux to cope with reduced CO₂ availability.

Chemical species carry information in two dimensions, in their concentrations and their isotopic signatures. The concentrations of metabolites or synthetic compounds describe the composition of a chemical or biological system, while isotopic signatures describe processes in the system by their reaction pathways, regulation, and responses to external stimuli. Stable isotopes are unique tracers of these processes because their natural abundances are modulated by isotope effects occurring in physical processes as well as in chemical reactions. Nuclear magnetic resonance (NMR) spectroscopy is a prime technique not only for identification and quantification of small molecules in complex systems but also for measuring intramolecular distribution of stable isotopes in metabolites and other small molecules. In this thesis, we use quantitative NMR in three fields: in food science, environmental pollutant tracing, and plant-climate science. The phospholipid (PL) composition of food samples is of high interest because of their nutritional value and technological properties. However, the analysis of PLs is difficult as they constitute only a small fraction of the total lipid contents in foods. Here, we developed a method to identify PLs and determine their composition in food samples, by combining a liquid-liquid extraction approach for enriching PLs, with specialized 31P,1H-COSY NMR experiments to identify and quantify PLs. Wide-spread pollution with synthetic compounds threatens the environment and human health. However, the fate of pollutants in the environment is often poorly understood. Using quantitative deuterium NMR spectroscopy, we showed for the nitrosamine NDMA and the pesticide DDT how intramolecular distributions (isotopomer patterns) of the heavy hydrogen isotope deuterium reveal mechanistic insight into transformation pathways of pollutants and organic compounds in general. Intramolecular isotope distributions can be used to trace a pollutant’s origin, to understand its environmental transformation pathways and to evaluate remediation approaches. The atmospheric CO2 concentration ([CO2]) is currently rising at an unprecedented rate and plant responses to this increase in [CO2] influence the global carbon cycle and will determine future plant productivity. To investigate long-term plant responses, we developed a method to elucidate metabolic fluxes from intramolecular deuterium distributions of metabolites that can be extracted from historic plant material. We show that the intramolecular deuterium distribution of plant glucose depends on growth [CO2] and reflects the magnitude of photorespiration, an important side reaction of photosynthesis. In historic plant samples, we observe that photorespiration decreased in annual crop plants and natural vegetation over the past century, with no observable acclimation, implying that photosynthesis increased. In tree-ring samples from all continents covering the past 60 – 700 years, we detected a significantly smaller decrease in photorespiration than expected. We conclude that the expected “CO2 fertilization” has occurred but was significantly less pronounced in trees, due to opposing effects. The presented applications show that intramolecular isotope distributions not only provide information about the origin and turnover of compounds but also about metabolic regulation. By extracting isotope distributions from archives of plant material, metabolic information can be obtained retrospectively, which allows studies over decades to millennia, timescales that are inaccessible with manipulation experiments.

Le climat alpin à plus de 2400 mètres d’altitude montre des fortes variations de température, des intensités lumineuses très élevées (3000 µmol photons m-2 s-1) qui sont connues pour générer un état de réduction importante de la chaine de transport des électrons photosynthétique. Le bon fonctionnement du processus photosynthétique est primordial pour les quelques espèces de plantes vasculaires qui sont présentes à l’étage alpin et qui doivent terminer leur cycle de vie lors d’une très courte période de végétation.Soldanella alpina et Ranunculus glacialis sont deux espèces inféodées aux étages alpin et nival. Dans leur site naturel de croissance nous avons mesuré des températures faibles (0.7°C) et fortes (37°C) sous des lumières supérieures à 2500 µmol photons m-2 s-1. Chez les espèces non-alpines ces conditions induisent la photoinhibition du PSII, ce qui est évité chez S. alpina et R. glacialis, par des mécanismes très différents. Les systèmes antioxydants et le quenching non photochimique sont particulièrement importants chez S. alpina. Chez Ranunculus glacialis, la photorespiration reste très importante et un contenu élevé en PTOX est décrit. Le rôle des antioxydants et de la PTOX dans la photoprotection des deux espèces ont été étudiés. Dans une partie de thèse, nous avons montré qu’une diminution de la capacité antioxydante par une diminution de la concentration en glutathion n’affecte pas la tolérance vis-à-vis de la photoinhibition à basse température. Dans une deuxième partie les résultats supposent qu’une surexpression de la PTOX chez le tabac augmente la photoinhibition à lumière forte par production des espèces réactives d’oxygène. En utilisant différentes conditions environnementales de croissance pour Ranunculus glacialis, nous avons pu montrer que l’expression de la PTOX est induite par des fortes lumières et non par des basses températures. Grâce à une approche associant mesures d’échanges gazeux et mesures de la fluorescence de la chlorophylle, nous avons montré qu’un flux d’électrons conséquent vers l’oxygène, indépendant de la photorespiration, corrélait avec la présence de la PTOX mais que l’activité de la PTOX sous des conditions qui permettent l’assimilation du CO2 et la photorespiration n’est pas maximale. Grâce à des mesures de fluorescence chlorophyllienne en présence de différents inhibiteurs photosynthétiques, nous avons pu montrer que l’importance de ce flux d’électrons vers l’oxygène corrèle avec la quantité de PTOX présente dans les feuilles, dans des conditions réductrices. Ces résultats nous ont amenés à conclure que chez Ranunculus glacialis, la PTOX peut prendre en charge un flux significatif d’électrons, éviter ainsi l’apparition d’un état réduit de la chaine de transfert photosynthétique, et protéger la plante vis-à-vis de la photoinhibition en agissant comme une valve de sécurité. Ces travaux permettent d’apporter des précisions sur un modèle original de photoprotection, qui a été l’objet de nombreuses controverses. / The alpine climate above 2400 meters altitude shows large variations in temperature and very important light intensity (3000 µmol photons m-2 s-1), which are known to generate a state of significant reduction in the photosynthetic electron transport chain. The proper functioning of the photosynthetic process is essential for vascular plants species that are present in this alpine environment and must complete their life cycle within a very short growing season.Soldanella alpina and Ranunculus glacialis are two species restricted to alpine and snow floors. In their natural growth environment we measured very low (0.7 ° C) and high temperature (37 ° C) under lights above 2500 µmol photons m-2 s-1. Among non-alpine species such conditions induce photoinhibition of PSII, which is avoided in S. alpina and R. glacialis, by very different mechanisms. Antioxidant systems and non-photochemical quenching are particularly important in S. alpina. In Ranunculus glacialis, photorespiration remains very important and a high content of PTOX is described. The roles of antioxidants and PTOX in photoprotection of both species were studied.In one part of the thesis, we showed that a decrease in antioxidant capacity by reducing the concentration of glutathione does not affect tolerance to low-temperature photoinhibition. In the second part the results imply that overexpression of PTOX in tobacco enhances photoinhibition by strong light to produce reactive oxygen species.Using different environmental conditions for Ranunculus glacialis growth, we showed that expression of the PTOX is induced by strong light, but not by low temperatures. With an approach combining gas exchange measurements and chlorophyll fluorescence measurements, we showed that an electron flow to oxygen, independent of photorespiration, correlated with the presence of PTOX. Through measures of chlorophyll fluorescence in the presence of various inhibitors photosynthetic, we could show that the importance of this electron flow to oxygen correlates with the amount of PTOX in the leaves, under reducing conditions. These results led us to conclude that in Ranunculus glacialis, the PTOX may support a significant flow of electrons, thus avoiding the appearance of a reduced state of the photosynthetic chain transfer, and protect the plant from photoinhibition, acting as a safety valve. These studies are discussed to help clarify a new pathway of photoprotection, which was the subject of much controversy.

Les voies métaboliques les plus importantes dans le contrôle du stress oxydant chez la plante restent à élucider. Celles liées au glutathion jouent un rôle important. Cependant, les réactions responsables de l'oxydation du glutathion (du GSH en GSSG) n'ont pas encore été clairement identifiées. L’analyse des données biochimiques, transcriptomiques et génétiques soulèvent des questions pour mieux comprendre comment la régulation redox liée au stress pourrait influer sur la signalisation hormonale chez les plantes. Par une approche de génétique inverse utilisant, notamment, le mutant photorespiratoire conditionnel cat2, nous avons étudié la réponse et l'importance fonctionnelle de trois voies potentielles, médiées par les glutathion S-transférases, les peroxirédoxines dépendant de la glutarédoxine et les déhydroascorbate réductases (DHARs) chez Arabidopsis. Ainsi, l'interaction entre les DHARs semble être nécessaire pour coupler les pools d'ascorbate et de glutathion lors d’un stress oxydant. En complément à l'approche expérimentale, une modélisation mécaniste a permis d'étudier la production de H2O2 et son métabolisme, en lien avec l'activité catalase et la voie ascorbate-glutathion. Le modèle révèle que la catalase et l'ascorbate peroxydase prennent en charge de concert le traitement de H2O2, y compris dans les conditions optimales de croissance. Nos simulations suggèrent que la disponibilité en NADPH peut déterminer l'oxydation du glutathion via la monodéshydroascorbate réductase. Nos résultats expérimentaux et le modèle cinétique valident que la sensibilité du statut du glutathion au stress oxydant constitue un senseur approprié des augmentations du H2O2. / The most important metabolic pathways in the control of oxidative stress remain to be elucidated in plants. Those linked to glutathione play an important role. However, the reactions responsible for its oxidation have not been clearly identified. Here, analysis based on available biochemical, transcriptomic and genetic data emphasized likely important questions to be elucidated for a full understanding of how stress-related redox regulation might impinge on phytohormone-related signaling pathways. Using a reverse genetics approach and the photorespiratory conditional cat2 mutant, we studied the response and functional importance of three potential routes for glutathione oxidation pathways mediated by glutathione S-transferases, glutaredoxin dependent peroxiredoxins, and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H2O2 metabolism. In addition to experimental work, modelling is another way to investigate H2O2 production and its metabolism related to catalase activity and ascorbate glutathione pathway. This approach led to major conclusions, that catalase and ascorbate peroxidase can share the load in H2O2 processing even in optimal growth conditions. Furthermore, simulations propose that NADPH availability may determine glutathione oxidation through its influence on monodehydroascorbate reduction. Taken together, experimental results and our kinetic model strengthen that the sensitivity of glutathione status to oxidative stress acts as a suitable sensor of increased H2O2.

Pour étudier le rôle de la mitochondrie dans la tolérance à la sécheresse, la réponse à la contrainte hydrique a été comparée entre une lignée sauvage (WT) et un mutant CMSII (Cytoplasmic Male Sterile) de Nicotiana sylvestris. Chez le mutant CMSII, le complexe I mitochondrial est absent et la respiration est assurée par les NAD(P)H déshydrogénases alternes et elle est maintenue à un niveau supérieur de l’ordre de 20 à 30% à celui du WT. La différence observée entre les plantes WT et CMSII met en jeu non seulement le fonctionnement mitochondrial, mais également le fonctionnement des chloroplastes. En effet, l’activité photosynthétique du mutant est plus faible que celui du WT et elle est corrélée avec une plus faible conductance stomatique (gs) et mésophyllienne (gm).Après l’arrêt de l’arrosage, on observe que le contenu relatif en eau (RWC) diminue plus lentement chez les feuilles du CMSII. Ceci n’était pas le résultat d’une plus petite surface de transpiration ou d’une masse racinaire d’absorption plus élevé puisque le rapport partie aérienne/racine et la surface foliaire totale ont été similaires au début de l’expérience chez les deux génotypes. De plus la mutation n’a pas induit des changements au niveau des paramètres hydriques (P0, PTLP, RWCTLP) ni au niveau de la densité stomatique. La tolérance des plantes CMSII a été le plus probablement la conséquence de sa plus faible transpiration en conditions bien hydratées et aux premiers jours de déshydratation et non pas d’une meilleure efficacité d’absorption de l’eau puisque le contenu en eau du sol reste plus élevé chez CMSII après l’arrêt de l’arrosage. La plus faible conductance stomatique chez le CMSII bien hydraté a été expliquée par sa plus faible conductance hydraulique. De plus, contrairement au WT, le niveau des acides aminés totaux diminue au cours de la déshydratation lorsque le contenu en protéines solubles augmente chez les feuilles du CMSII, suggérant une accélération de la remobilisation des acides aminés. D’autre part, il a été aussi montré que le mutant CMSII est capable de s’acclimater mieux à la sécheresse que le WT lorsqu’ils ont été maintenus à un RWC de 80 % sur plusieurs jours. Sous ces conditions, la photosynthèse reste plus élevée chez le mutant que chez le WT. Cette meilleure acclimatation corrèle avec une plus forte photorespiration du CMSII sous conditions bien hydratées et sous conditions d’acclimatation. La photorespiration chez CMSII et le WT a été estimée par le transport électronique dévolu à l’oxygénation de RuBP et en plus par l’accumulation des métabolites impliqués dans la photorespiration. D’une part, l’acclimatation à la sécheresse diminue gm plus fortement chez le WT que chez le CMSII. D’autre part, le WT accumule la glycine ce qui laisse supposer que le glycine décarboxylase mitochondrial est plus affectée chez le WT que chez le CMS et inhibe ainsi la photorespiration. En effet, cette plus faible photorespiration chez le WT affecte les réactions primaires de la photosynthèse par une accumulation d’un gradient de protons estimé par le quenching non-photochimique (NPQ) de la fluorescence chlorophyllienne ce qui induit une diminution du transport électronique des réactions primaires de la photosynthèse. / To investigate the role of mitochondria in drought stress, the response to water deprivation was compared between Nicotiana sylvestris wild type (WT) plants and the CMSII respiratory complex I mutant. In CMSII, alternative NAD(P)H-dehydrogenases bypassing complex 1 allow respiration.. The difference of mitochondrial function between WT and CMSII plants affect also photosynthesis. The CMSII has lower photosynthetic actitvity than the WT and lower stomatal (gs) and internal (gm) conductances to CO2. When watering of plants with similar leaf surface and similar shoot/root ratio was stopped the relative water content (RWC) declined faster in WT as compared to CMSII leaves. Furthermore, CMSII and WT leaves had the same osmotic potential at leaf saturation (P0) and at leaf turgor lost pressure (PTLP) and the same stomatal density. The slower decline of RWC in CMSII, compared to WT leaves, was most likely the consequence of the lower stomatal conductance (gs) under well-watered conditions and during the first days after withholding watering, The lower stomatal conductance of well-watered CMSII leaves correlated with a lower hydraulic conductance of leaves. Remarkably, total free amino acid levels declined and total soluble protein content increased in CMSII leaves, while the opposite was observed in WT leaves. This suggests protein synthesis in CMSII but protein degradation in WT leaves during drought stress. We also show that CMSII leaves better acclimate to drought stress than the WT leaves. After several days at 80 % RWC , photosynthesis is higher in the mutant than in WT. As compared to the WT, the mutant shows higher rates of photorespiration before and after acclimation to drought.The strong accumulation of glycine in the WT suggests that photorespiration may be limited at the level of glycine decarboxylase. In addition, after acclimation to drought gm declined markedly in WT but not in CMSII leaves, thus further limiting CO2 supply for photosynthesis in the WT. The resulting lower photosynthesis and photorespiration in WT leaves affect also the primary reaction of photosynthesis by increasing the non-photochemical fluorescence quenching (NPQ) and decreasing linear electron transport.

In the natural environment plants are confronted to a multitude of biotic and abiotic stress factors that must be perceived, transduced, integrated and signaled in order to achieve a successful acclimation that will secure survival and reproduction. Plants have to deal with excess excitation energy (EEE) when the amount of absorbed light energy is exceeding that needed for photosynthetic CO2 assimilation. EEE results in ROS formation and can be enhanced in low light intensities by changes in other environmental factors. The lesions simulating disease resistance (lsd1) mutant of Arabidopsis spontaneously initiates spreading lesions paralleled by ROS production in long day photoperiod and after application of salicylic acid (SA) and SA-analogues that trigger systemic acquired resistance (SAR). Moreover, the mutant fails to limit the boundaries of hypersensitive cell death (HR) after avirulent pathogen infection giving rise to the runaway cell death (rcd) phenotype. This ROS-dependent phenotype pointed towards a putative involvement of the ROS produced during photosynthesis in the initiation and spreading of the lesions. We report here that the rcd has a ROS-concentration dependent phenotype and that the light-triggered rcd is depending on the redox-state of the PQ pool in the chloroplast. Moreover, the lower stomatal conductance and catalase activity in the mutant suggested LSD1 was required for optimal gas exchange and ROS scavenging during EEE. Through this regulation, LSD1 can influence the effectiveness of photorespiration in dissipating EEE. Moreover, low and high SA levels are strictly correlated to lower and higher foliar H2O2 content, respectively. This implies an essential role of SA in regulating the redox homeostasis of the cell and suggests that SA could trigger rcd in lsd1 by inducing H2O2 production. LSD1 has been postulated to be a negative regulator of cell death acting as a ROS rheostat. Above a certain threshold, the pro-death pathway would operate leading to PCD. Our data suggest that LSD1 may be subjected to a turnover, enhanced in an oxidizing milieu and slowed down in a reducing environment that could reflect this ROS rheostat property. Finally, the two protein disulphide isomerase boxes (CGHC) present in the protein and the down regulation of the NADPH thioredoxin reductase (NTR) in the mutant connect the rcd to a putative impairment in the reduction of the cytosolic thioredoxin system. We propose that LSD1 suppresses the cell death processes through its control of the oxidation-reduction state of the TRX pool. An integrated model considers the role of LSD1 in both light acclimatory processes and in restricting pathogen-induced cell death.

Ribulose-1,5-Bifsfosfato (RuBP) carboxilase/oxigenase (RuBisCO) é a enzima chave para a fixação do carbono atmosférico e para a produtividade das plantas. Não há, até o momento, uma metodologia estabelecida para otimizar o processo de fixação do CO2 nas diferentes espécies de plantas. Entretanto, a disponibilidade de um protocolo de transformação genética de cloroplasto de tabaco permite tentativas de manipulação da enzima RuBisCO visando aumento da eficiência fotossintética. Nas plantas, esta proteína é formada por 8 subunidades menores codificadas pelo gene rbcS localizado no genoma nuclear e por 8 subunidades maiores codificadas pelo gene rbcL localizado no genoma de cloroplastos. Neste trabalho, dois genes rbcL-sintéticos, um com a substituição da alanina (A) 378 por uma valina (V) (A378V) e outro sem a substituição foram utilizados para a construção dos vetores pTT629, pTT630, pTT632 e pTT633. Estes vetores foram usados para transformar o cloroplasto de folhas de tabaco, pelo método de biolística. Um total de 35 plantas transplastômicas se desenvolveram sob seleção dos antibióticos espectinomicina (500 mg/L) e estreptomicina (500 mg/L) e a análise molecular dos sítios de restrição AccI, EcoRI, NdeI e NsiI, de fragmentos amplificados da sequência codante atpB::rbcL:: aadA:: accD demonstrou a integração dos genes rbcL-sintéticos em 11 linhagens transplastômicas. Sementes F1 destas plantas demonstraram ser homoplásmicas pela germinação na presença do antibiótico espectinomicina (500 mg/L). Análises fisiológicas das taxas de fotossíntese (A), condutância estomática (gs) e de transpiração (E) das plantas transplastômicas (A378V) mantidas em casa-de-vegetação produziram valores maiores e significativos, quando comparados com as plantas sem a mutação e controle não transgênicos. O aumento da taxa de fotossíntese das linhagens transplastômicas indicam a possibilidade de aumento da atividade catalítica da RuBisCO. A compreensão da interação fotossintética com a atividade fotorrespiratória poderá permitir explorar e estender possíveis benefícios, como o aumento da produtividade em cultivares de interesse agronômico. / Ribulose-1,5-Bifsfosfato (RuBP) carboxylase/ oxygenase (RuBisCO) is the key enzyme for the fixation of atmospheric carbon and productivity of plants. At moment, no single solution to optimize the CO2 fixing process by the different species of plants. The availability of a few efficient chloroplast transformation protocols for all cultivars also directs attempts to manipulate the larger and small subunit of RuBisCO. In plants, this protein consists of coding form eight smaller subunits encoding the rbcS gene and 8 larger subunits of the rbcL gene respectively located in the nucleus and chloroplasts. Using two rbcL-synthetic genes, with an alanine (Ala) 378 substituting a valine (Val) (A378V) and another one without the replacement were used in the construction of pTT629, pTT630, pTT632 and pTT633 vectors, which were used in the method of biolistic to driving these transgenes into the chloroplast genome of tobacco. A total of 35 transplastomic plants were grown under selection of antibiotics spectinomycin (500mg/ L) and streptomycin (500mg/ L) and the molecular analysis using restriction sites AccI, EcoRI, NdeI and NsiI from the amplified fragments of atpB::rbcL:: aadA:: accD sequence displayed the rbcL-synthetic genes integrated into the plastome of the 11 transplastomic lines. The F1 seeds of these plants were shown to be homoplasmic from germinating in the presence of the antibiotic spectinomycin (500mg / L). The physiology analyzes of photosynthesis (A), stomatal conductance (gs) and transpiration (E) rates of these transplastomic lines (A378V) plants kept in green-house produced the highest and significant values when when compared to the control plants without the mutation and non-transgenic control. The increase of the photosynthesis rate form transplastomic lines indicates the possibility of increasing the catalytic activity of RuBisCO. The understanding of the photosynthetic interaction with photorespiration activity may allow explore more the potential benefits, such as increased productivity in crops of agronomic interest.

<p>In the natural environment plants are confronted to a multitude of biotic and abiotic stress factors that must be perceived, transduced, integrated and signaled in order to achieve a successful acclimation that will secure survival and reproduction. Plants have to deal with excess excitation energy (EEE) when the amount of absorbed light energy is exceeding that needed for photosynthetic CO2 assimilation. EEE results in ROS formation and can be enhanced in low light intensities by changes in other environmental factors.</p><p>The lesions simulating disease resistance (lsd1) mutant of Arabidopsis spontaneously initiates spreading lesions paralleled by ROS production in long day photoperiod and after application of salicylic acid (SA) and SA-analogues that trigger systemic acquired resistance (SAR). Moreover, the mutant fails to limit the boundaries of hypersensitive cell death (HR) after avirulent pathogen infection giving rise to the runaway cell death (rcd) phenotype. This ROS-dependent phenotype pointed towards a putative involvement of the ROS produced during photosynthesis in the initiation and spreading of the lesions.</p><p>We report here that the rcd has a ROS-concentration dependent phenotype and that the light-triggered rcd is depending on the redox-state of the PQ pool in the chloroplast. Moreover, the lower stomatal conductance and catalase activity in the mutant suggested LSD1 was required for optimal gas exchange and ROS scavenging during EEE. Through this regulation, LSD1 can influence the effectiveness of photorespiration in dissipating EEE. Moreover, low and high SA levels are strictly correlated to lower and higher foliar H2O2 content, respectively. This implies an essential role of SA in regulating the redox homeostasis of the cell and suggests that SA could trigger rcd in lsd1 by inducing H2O2 production.</p><p>LSD1 has been postulated to be a negative regulator of cell death acting as a ROS rheostat. Above a certain threshold, the pro-death pathway would operate leading to PCD. Our data suggest that LSD1 may be subjected to a turnover, enhanced in an oxidizing milieu and slowed down in a reducing environment that could reflect this ROS rheostat property. Finally, the two protein disulphide isomerase boxes (CGHC) present in the protein and the down regulation of the NADPH thioredoxin reductase (NTR) in the mutant connect the rcd to a putative impairment in the reduction of the cytosolic thioredoxin system. We propose that LSD1 suppresses the cell death processes through its control of the oxidation-reduction state of the TRX pool. An integrated model considers the role of LSD1 in both light acclimatory processes and in restricting pathogen-induced cell death.</p>

Climate is changing and it is certain that this change is due to human activities. Atmospheric greenhouse gases have been rising in an unprecedented way during the last two centuries, although the land biosphere has dampened their increase by absorbing CO2 emitted by anthropogenic activities. However, it is unclear if this will continue in the future. This uncertainty makes it difficult to predict future climate changes and to determine how much greenhouse gas emissions must be reduced to protect climate. To understand the future role of plants in limiting the atmospheric CO2 level, the effect of increasing CO2 on plant photosynthesis and productivity has been studied. However, studies on trees showed contradictory results, which depended on the duration of the experiment. This revealed that an initial strong CO2 fertilization may be a transient response that disappears after a few years. Because climate changes over centuries, we must explore the response of vegetation to increasing CO2 on this time scale. Studying tree rings is a good alternative to impractical decade-long experiments, because trees have experienced the CO2 increase during the last 200 years and may already have responded to it. This thesis shows that the intramolecular distribution of the stable hydrogen isotope deuterium (deuterium isotopomer distribution, DID) of tree rings is a reliable tool to study long-term plant-climate adaptations. The premise for this is that the deuterium abundance in tree rings depends on environmental as well as physiological factors. Using newly developed methodology for DID measurements, the influences of both factors can be separated. Applied to tree rings, separating both factors opens a strategy for simultaneous reconstruction of climate and of physiological responses. The results presented show that DIDs are influenced by kinetic isotope effects of enzymes, allowing studies of metabolic regulation. We show that the abundances of specific D isotopomers in tree-ring cellulose indeed allow identifying environmental and physiological factors. For example, the D2 isotopomer is mostly influenced by environment, its abundance should allow better reconstruction of past temperature. On the other hand, the abundance ratio of two isotopomers (D6R and D6S) depends on atmospheric CO2, and might serve as a measure of the efficiency of photosynthesis (ratio of photorespiration to assimilation). The presence of this dependence in all species tested and in tree-ring cellulose allows studying adaptations of plants to increasing CO2 on long time scales, using tree-ring series or other remnant plant material. / Klimatet förändras och det är numera allmänt vedertaget att detta beror på människans aktiviteter. Halten av växthusgaser har stigit onormalt mycket under de senaste två århundradena och detta beror i största del på människans användning av fossila bränslen. Landbiosfären har hittills haft en buffrande effekt på klimatförändringen eftersom den tar upp och lagrar mycket av växthusgasen CO2. Det är dock osäkert om, och i så fall hur länge, denna effekt kvarstår. Detta gör det mycket svårt att förutsäga framtida klimatförändringar, och därmed hur mycket utsläppen av växthusgaser måste reduceras för att skydda klimatet. För att förstå växternas framtida förmåga att begränsa halten atmosfäriskt CO2 har man studerat effekten av förhöjda halter av CO2 på växters fotosyntes och produktivitet. Resultaten av dessa försök varierar i stor omfattning. Studier på träd odlade under höga halter CO2 indikerar att den initiala ökningen av en trädets produktivitet kan vara en temporär effekt som försvinner redan efter några år. Eftersom klimatförändringen sker under århundraden, måste även växternas anpassningar på förhöjd CO2 halt utforskas på denna tidsskala, men experiment som skulle ta tiotals år är opraktiska att utföra. Trädringar är ett bra sätt att studera sådana anpassningar, eftersom träd redan har upplevt de senaste två hundra årens ökning av koldioxid och dess trädringar därför kan innehålla information om en respons som redan skett. Denna avhandling visar att den intramolekylära fördelningen av den stabila väteisotopen deuterium i trädringar är en tillförlitlig metod för att studera växters anpassningsförmåga till långsiktiga klimatförändringar. Antagandet bakom denna strategi är att isotopfördelningen i trädringar beror på faktorer både från miljön och växtens fysiologi. Om båda faktorerna skulle kunna utvinnas från trädringar, skulle detta öppna en helt ny väg för parallell rekonstruktion av klimatet och växters anpassning till det. Avhandlingen presenterar den första tekniken för att mäta isotopfördelningen av deuterium i växtglukos. Resultaten visar att deuteriumfördelningen hos växtglukos påverkas av enzymers isotopeffekter, vilket möjliggör att regleringen av växternas metabolism kan kartläggas. I avhandlingen bevisas att halten deuterium i skilda intramolekylära positioner (isotopomerer) av glukos från trädringcellulosa bestäms av miljöfaktorer respektive trädets fysiologi. T.ex. påverkas deuteriumhalten i position 2 (D2 isotopomer) av glukosmolekylen huvudsakligen av miljön, vilket kan användas för att förbättra temperaturrekonstruktioner från trädringar. Å andra sidan är kvoten deuterium mellan två andra positioner (D6R och D6S) relaterat till halten atmosfäriskt koldioxid, och kvoten skulle kunna användas som mått för fotosyntesens effektivitet, dvs. förhållandet mellan fotorespiration och fotosyntes. Närvaron av denna relation i trädringar och annat växtmaterial i alla de växter vi hittills studerat, öppnar en helt ny möjlighet att studera växters anpassning till den ökande mängden CO2 i atmosfären under århundraden.

SOUSA, Rachel Hellen Vieira de. Silenciamento de peroxidase do ascorbato peroxissomal induz mudanças antioxidantes capazes de atenuar estresse oxidativo induzido por excesso de H2O2 na deficiência de catalase. 2014. 93 f. Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza-CE, 2014. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-06-29T12:54:56Z No. of bitstreams: 1 2014_dis_rhvsousa.pdf: 1853718 bytes, checksum: da11921f6237d61d7d067fa8e43834c7 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:13:09Z (GMT) No. of bitstreams: 1 2014_dis_rhvsousa.pdf: 1853718 bytes, checksum: da11921f6237d61d7d067fa8e43834c7 (MD5) / Made available in DSpace on 2016-08-02T20:13:09Z (GMT). No. of bitstreams: 1 2014_dis_rhvsousa.pdf: 1853718 bytes, checksum: da11921f6237d61d7d067fa8e43834c7 (MD5) Previous issue date: 2014 / The ascorbate peroxidase (APX) and catalase (CAT) are the most important enzymes in removing H2O2 of plant cells. Both are present in peroxisomes. In high photorespiration, the amount of H2O2 in peroxisomes is increased, due to greater activity of glycolate oxidase (GO), enzyme producer of H2O2. Catalase has greater involvement in the removal of H2O2 presenting a high Km and the ability to scavenge higher concentrations of H2O2 than APX. The importance of the peroxisomal isoform of APX in antioxidant metabolism of plant cells is still unknown. There are few papers in literature reporting the role of pAPX. Thus, we performed this work with the objective of evaluating the effect of catalase inhibition in rice plants (APX4) silenced in APX’s peroxisomal isoform. Initially, it was performed a characterization work of three lines of APX4 (Lg, Lh and Lj), aiming to select one line for future studies. It was observed lower GO activity and a slight increase in net photosynthesis in the three mutant lines. Based on biochemical, physiological and photosynthetic parameters, APX4-Lg line was chosen. The APX4 silencing resulted in suppression of expression of the other peroxisomal isoform, APX3. Subsequently, experiments were conducted with catalase inhibition by aminotriazole (AT) in plants NT and APX4. Mutant plants had less electrolyte leakage than NT plants when catalase was inhibited. The synthesis of GSH in the absence of CAT was higher in NT plants. After CAT inhibition, GPX activity increase was higher in APX4 than in NT plants. In order to induce a greater effect of the CAT absence, an experiment was performed combining CAT inhibition with light (1000 μmol photons m -2 s -1), in leaf segments. Similar to the results found in plants, APX4 suffered less than NT plants, with catalase inhibition. APX4 plants also showed higher Fv/Fm, lower electrolyte leakage and lower accumulation of H2O2, in AT+light treatment. Experiments with inhibition of GO were also conducted in order to reduce photorespiration. These results show that plants silenced in APX4 exhibited greater tolerance to inhibition of catalase by a new redox homeostasis able to cope with the catalase inhibition. Further studies are needed to elucidate the mechanisms that APX4 plants have developed to provide better acclimation to catalase inhibition. / As enzimas Peroxidase do ascorbato (APX) e catalase (CAT) são as mais importantes na remoção de H2O2 nas células vegetais. Ambas estão presentes nos peroxissomos. Em elevada fotorrespiração a quantidade de H2O2 nos peroxissomos é aumentada, em função de uma maior atividade de glicolato oxidase (GO), enzima produtora de H2O2. A catalase se destaca na remoção de H2O2 por possuir um Km elevado e ser capaz de eliminar maiores concentrações de H2O2 do que a APX. A importância da isoforma peroxissomal da APX no metabolismo antioxidante das células vegetais ainda é desconhecida. Há poucos trabalhos na literatura que estudam a papel da APXp. Para tanto, realizamos esse trabalho com o objetivo de avaliar o efeito da inibição da catalase em plantas de arroz (APX4) silenciadas em uma isoforma peroxissomal de APX. Inicialmente foi realizada uma caracterização de três linhagens de APX4 (Lg, Lh e Lj), com o objetivo de selecionar uma para estudos futuros. Foi observado uma menor atividade de GO e um leve aumento na fotossíntese liquida nas três linhagens mutantes. Com base em parâmetros bioquímicos, fisiológicos e fotossintéticos, foi escolhida a linhagem APX4-Lg. O silenciamento da APX4 resultou em supressão da expressão da outra isoforma peroxissomal, APX3. Posteriormente, foram realizados experimentos inibindo a catalase com aminotriazol (AT) em plantas NT (não transformadas) e APX4. As plantas mutantes tiveram um menor vazamento de eletrólitos do que as NT, com a inibição da catalase. A síntese de GSH (glutationa reduzida), na ausência de CAT, foi maior nas plantas NT. Com a inibição da CAT a atividade de GPX (peroxidade da glutationa) aumentou mais nas plantas APX4. Com o objetivo de induzir uma maior efeito da ausência de CAT, foi realizado um experimento combinando inibição CAT com luz (1000 μmol fótons m-2s-1), em segmentos. Semelhante ao resultados encontrados em plantas, as plantas APX4 sofreram menos com a inibição da catalase. Visto que no tratamento de AT+luz as plantas mutantes, comparadas com as NT, apresentaram maior Fv/Fm, menor vazamento de eletrólitos e menor acumulação de H2O2. Experimentos com inibição de GO também foram conduzidos, com o intuito de reduzir a fotorrespiração. Estes resultados mostram que as plantas silenciadas em APX4 apresentam uma maior tolerância a inibição da catalase através de uma nova homeostase redox capaz de lidar melhor com ausência de catalase. Estudos posteriores são necessários para elucidar quais mecanismos as plantas APX4 desenvolveram para conferir a elas uma melhor aclimatação a ausência de catalase.